Compositions and methods for increasing salt tolerance in plants

ABSTRACT

Methods and compositions for increasing tolerance to abiotic stresses in plants are provided. One embodiment provides a method for increasing salt tolerance in a plant by treating the plant with an amount of 2-keto-4-methylthiobutyric acid (KMBA) effective to increase salt tolerance in the plant relative to an untreated plant. Typically KMBA is in an aqueous solution used to irrigate the plant, seed, or seedling. In a preferred embodiment, the aqueous solution is a salt solution, including but not limited to seawater. The KMBA is typically present in the aqueous solution at 1.0 to 250 nM, preferably at least 100 nM.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit and priority of U.S. ProvisionalPatent Application No. 62/630,013 filed on Feb. 13, 2018, and is acontinuation-in part of U.S. Ser. No. 16/072,137 filed on Jul. 23, 2018,which is a National Phase application under 35 U.S.C. § 371 ofInternational Application No. PCT/IB2017/050314, filed on Jan. 20, 2017,entitled “COMPOSITIONS AND METHODS FOR PROVIDING PLANTS WITH TOLERANCETO ABIOTIC STRESS CONDITIONS”, which claims benefit of and priority toU.S. Provisional Patent Application No. 62/281,404 filed on Jan. 21,2016, and are incorporated herein in their entirety.

FIELD OF THE INVENTION

The invention is generally directed to plant cultivation under abioticstress conditions.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Jul. 27, 2018, as a text file named“KAUST_2018_114_02_ST25.txt” created on Jul. 27, 2018, and having a sizeof U.S. Pat. No. 5,507,846 bytes is hereby incorporated by referencepursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND OF THE INVENTION

Plants as sessile organisms are facing multiple stresses during theirlifetime. Among them, abiotic stresses, such as salt stress, can causesevere crop yield reduction, leading to food security issues in manyregions of the world. In order to respond to growing food demands,especially in the context of global climate change and an increasingworld population, it then becomes urgent to develop new strategies togenerate more tolerant crops to abiotic stresses.

Therefore it is an object of the invention to provide compositions andmethods for increasing tolerance in plants to abiotic stresses.

It is another object of the invention to provide seed coatingcompositions that provide plants with resistance or tolerance to abioticstress conditions.

It is still another object to provide methods for growing plants underabiotic stress conditions.

SUMMARY OF THE INVENTION

Methods and compositions for increasing tolerance to abiotic stresses inplants are provided. The methods include providing the plant with2-keto-4-methylthiobutyric acid (KMBA) exogenously (“exogenous KMBAmethod”), alone or in combination with modifying the plant with amicrobial system that results in production of KMBA in the plant(“endogenous KMBA method”). The exogenous KMBA method include treatingseeds, seedlings or mature plants with 2-keto-4-methylthiobutyric acid(KMBA). The exogenous KMBA method in one increases salt tolerance in aplant by treating the plant with an amount of KMBA) effective toincrease salt tolerance in the plant relative to an untreated plant.Typically KMBA is in an aqueous solution used to irrigate the plant,seed, or seedling. In a preferred embodiment, the aqueous solution is asalt solution, including but not limited to seawater. The KMBA istypically present in the aqueous solution at 1.0 to 250 nM, preferablyat least 100 nM.

Another embodiment provides cultivating a plant in sodic soil byirrigating the plant, seed, or seedling in the sodic soil with a aqueoussolution containing 1.0 to 250 nM KMBA. The aqueous solution istypically water. The water can be rainwater, wellwater, or water fromnatural sources such as rain, lakes, glaciers, rivers, and streams.

When KMBA is combined with a salt solution, the salt is can be from 1.0to 10% (w/v) salt. In a preferred embodiment, the salt solution is about3.5% salt. The salt solution can contain salts of sodium, chloride,sulfates, magnesium, calcium, potassium, and combinations thereof.

Another embodiment provides a method for cultivating a plant by plantingseeds or seedlings of the plant and irrigating the seeds or seedlingswith a saline solution comprising KMBA. The plant can be an agriculturalcrop plant or a forage crop plant. Representative plants include but arenot limited to rice, wheat, sugarcane, maize, soybean, cotton,vegetables, rape, mustard, sorghum, millet, grass, Brassica spp.,rapeseed, barley, hay, and alfalfa.

Still another embodiment provides a method of increasing tolerance toabiotic stress in a plant by treating the plant with a compositioncontaining an effective amount of KMBA to increase tolerance to theabiotic stress. In certain embodiments, the abiotic stress is due towater salinity, soil salinity, or both. The KMBA can be present at aconcentration of 1.0 to 250 nM, preferably 100 nM.

Yet another embodiment provides an irrigation solution containing 1.0 to250 nM KMBA in an aqueous saline solution. The aqueous saline solutiontypically contains 1 to 10% salt (w/v). The aqueous solution ispreferably seawater.

The endgenous KMBA method providing seeds, seedlings or mature plantswith a microbial system that results in production of KMBA in matureplants. In some embodiments, seeds or plant roots can be innotculatedwith or coated with a microbial system that results in production ofKMBA in mature plants. A preferred microbial system Enterobacter sp.SA187. In other embodiments, seeds, seedling and plants can be grownusing substrates containing an effective amount of Enterobacter sp.SA187 to inhibit or reduce abiotic stress in the plant. Suitable plantsubstrates include, but are not limited to soil, peat, compost,vermiculite, perlite, sand, clay and combinations thereof.

Another embodiment provides a bacterium genetically engineered toexpress KMBA or increased levels of KMBA relative to an unmodifiedbacterium, which can be used as a a microbial system that results inproduction of KMBA in mature plants. In a preferred embodiment thebacterium is an endophytic bacterium. The engineered bacterium can beused to inoculate plants and increase tolerance in the plant to abioticstress conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show growth parameters of alfalfa in field trials. FIGS. 1Aand 1D are bar graphs showing plant height (cm) of alfalfa plantsinoculated with mock (gray bar) and SA187 (green bar) and subjected tolow or high saline conditions. FIGS. 1B and 1E are bar graphs showingfresh weight (tons/ha) of alfalfa plants inoculated with mock (gray bar)and SA187 (green bar) and subjected to low or high saline conditions.FIGS. 1C and 1F are bar graphs showing dry weight (tons/ha) of alfalfaplants inoculated with mock (gray bar) and SA187 (green bar) andsubjected to low and high saline conditions. Data from the first season(FIGS. 1A, 1B, 1C) and second season (FIGS. 1D, 1E, 1F) are displayed;each column represents a mean of 3 harvests from 4 experimental plotserror bars are SE. An increase of each parameter for SA187-treatedplants related to Mock is indicated in %. Asterisks indicate astatistical difference (*P<0.05;**P<0.01,***P<0.001).

FIGS. 2A-2J show that SA187 enhances Arabidopsis tolerance to saltstress. FIG. 2A is a bar graph that shows germination efficiency on ½ MSmedium without (mock) or with SA187 (+SA187) (n>300, 3 biologicalreplicates, bars represent SE). FIG. 2B is a bar graph that showsaverage root hair length (μm) of 10% longest root hairs (n>70) in5-day-old seedlings grown vertically on ½ MS medium without (mock) orwith SA187. Error bars represent SE. FIG. 2C is a representative imageshowing typical root hair morphology of 5-day-old seedlings used for theanalysis in FIG. 2B. Scale bar represents 200 μm. FIG. 2D is a linegraph showing root length (cm) over time in SA187-inoculated (uppersolid line) or mock-treated (lower solid line) Arabidopsis seedlingsafter transfer of 5-day-old seedlings from ½ to ½ MS with or without 100mM NaCl (dashed lines) (n=60). Error bars represent SE. FIG. 2E arerepresentative images of SA187-colonized 17-day-old plants showingenhanced growth under salt stress (½ MS+100 mM NaCl) but negligibledifferences under normal conditions (½ MS). Scale bar represents 1 cm.FIGS. 2F-2J are bar graphs showing (FIG. 2F) total plant dry weight(mg), (FIG. 2G) total plant fresh weight (mg), (FIG. 2H) shoot freshweight (mg), and (I) root fresh weight (mg) of 17-day-old seedlings and(FIG. 2J) lateral root density of 13-day-old seedlings inoculated bySA187 (green bar) or mock-treated (gray bar) transferred 5 days aftergermination from½ MS to ½ MS with or without 100 mM NaCl. All plotsrepresent the mean of three biological replicates (n>39). Error barsrepresent SE. Asterisks indicate a statistical difference based on theStudent t-test (*P<0.05;**P<0.01;***P<0.001).

FIGS. 3A-3F show ion content in Arabidopsis seedlings. FIGS. 3A-3C arebar graphs showing (FIG. 3A) shoot Na+ content, (FIG. 3B) shoot K+content, and (FIG. 3C) shoot Na+/K+ ratio of 17-day-old mock-(gray bar)or SA187-(green bar) inoculated Arabidopsis seedlings exposed for 12days to ½ MS with or without 100 mM NaCl {48>n>36). FIGS. 3D-3F are bargraphs showing (FIG. 3D) root Na+ content, (FIG. 3E) root K+ content,and (FIG. 3F) root Na+/K+ ratio of 17-day-old mock-(gray bar) orSA187-(green bar) inoculated Arabidopsis seedlings exposed for 12 daysto ½ MS with or without 100 mM NaCl (48>n>12). All plots represent themean of three biological replicates, and error bars represent SE.Asterisks indicate a statistical difference based on Mann-Whitney test(*P<0.05;**P<0.01,***P<0.001).

FIGS. 4A-4F shows transcriptome analysis of Arabidopsis response toSA187. FIG. 4A is a heat map showing response of genes in Arabidopsisseedlings in response to SA187, salt (100 mM NaCl) or both treatmentsbased on the RNA-Seq analysis. Heat map colors indicate expressionlevels. FIGS. 4B-4F are hierarchical clustering of up-and down-regulatedgenes from the RNA-Seq analysis. For every gene, FPKM values werenormalized. For the most relevant clusters, gene families significantlyenriched are indicated based on gene ontology.

FIGS. 5A-5C are bar graphs showing (FIG. 5A) Salicylic acid (SA), (FIG.5B) abscisic acid (ABA) and (FIG. 5C) jasmonic acid (JA) content ofmock- and SA187-inoculated plants after growth on ½ MS with or without100 mM NaCl (salt) for 12 days. Error bars indicate SE based on threebiological replicates. Asterisks indicate a statistical difference basedon Mann-Whitney test (*P<0.05). FIGS. 5D-5E are representative photosshowing the ethylene reporter, pEBF2::GUS, in primary root tips of (FIG.5D) mock- and (FIG. 5E) SA187-inoculated, and (FIG. 5F) ACC-treated7-day-old seedlings under normal conditions (salt stress conditionsprovided similar results). Scale bar=100 μm.

FIGS. 6A and 6B are bar graphs showing (FIG. 6A) the fresh weight (mg)and (FIG. 6B) beneficial index (fresh weight ratio betweenSA187-colonized and control seedlings) of mock-or SA187-inoculatedmutants in hormonal pathways transferred to ½ MS+100 mM NaCl for 12days. acs=heptuple mutant acsl-1 acs2-1 acs4-1 acsS-2 acs6-1 acsl-1acs9-1, and pyrl/pyl =quadruple mutant pyrl py/1 py/2 py/4. All plotsrepresent the mean of three biological replicates (n>36). FIG. 6C is agraph showing Arabidopsis growth parameters under salt stress. 100 nMACC partially mimics the effect of SA187 on salt stress toleranceimprovement in Arabidopsis seedlings. Five-day-old-seedlings weretransferred to ½ MS+ 100 mM NaCl with or without ACC and evaluated after12 additional days. SA187-inoculated plants were used for comparison.FIGS. 6D-6G are bar graphs showing qPCR expression analysis of fourethylene-associated genes, (FIG. 6D) ERF105 (At5g51190), (FIG. 6E) RAV1(At1g13260), (FIG. 6F) ERF018 (At1g74930), and (FIG. 6G) SZF1(At3g55980), in 17-day-old mock-(gray bar) and SA187-(green bar)inoculated Arabidopsis seedlings exposed for 12 days to ½ MS with orwithout 100 mM NaCl. Normalized expression indicates the linear foldchange compared to mock-treated plants on ½ MS. Values represent meansof three biological experiments, each in three technical replicates.Error bars indicate SE. FIG. 6H is a bar graph showing total freshweight of mock-(gray bars) and SA187-(green bars) inoculated 18-day-oldArabidopsis seedlings on ½ MS with 100 mM NaCl supplemented with theethylene synthesis inhibitor AVG (1 μm) or ethylene signaling inhibitorAgN03 (1 μm). Error bars representing SE and beneficial index (%) aredisplayed. Asterisks indicate a statistical difference based on Studentt-test (*P<0.05;**P<0.01;***P<0.001).

FIGS. 7A and 7B are bar graphs showing qPCR analysis of the methioninesalvage cycle gene expression of SA187 colonizing plants in (FIG. 7A)control or (FIG. 7B) salt stress conditions compared to SA187 alone in ½MS with or without 100 mM NaCl. Values represent means of threebiological experiments, each in three technical replicates. Error barsindicate SE. FIG. 7C is a graph showing Arabidopsis growth parametersunder salt stress. Mock plants were transferred 5 days after germinationto ½ MS+ 100 mM NaCl with or without KMBA and evaluated after 12additional days. SA187-inoculated plants transferred to ½ MS+ 100 mMNaCl were used as a positive control. FIGS. 7D and 7E are bar graphsshowing total fresh weight of mock-(gray bars) and SA187-(green bars)inoculated 17-day-old Arabidopsis seedlings grown on ½ MS medium (FIG.7D) or ½ MS with 100 mM NaCl (FIG. 7E) supplemented with 3 μM DNPH. Allplots represent the mean of four biological replicates (n>75). Errorbars representing SE, beneficial index (%) is displayed above. Asterisksindicate a statistical difference based on Student t-test (***P<0.001).FIG. 7F shows the methionine salvage pathway.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The use of the terms “a,” “an,” “the,” and similar referents in thecontext of describing the presently claimed invention (especially in thecontext of the claims) are to be construed to cover both the singularand the plural, unless otherwise indicated herein or clearlycontradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Use of the term “about” is intended todescribe values either above or below the stated value in a range ofapprox. +/−10%; in other embodiments the values may range in valueeither above or below the stated value in a range of approx. +/−5%; inother embodiments the values may range in value either above or belowthe stated value in a range of approx. +/−2%; in other embodiments thevalues may range in value either above or below the stated value in arange of approx. +/−1%. The preceding ranges are intended to be madeclear by context, and no further limitation is implied. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

The term “plant” refers to the entire plant, seeds of the plant, andseedlings of the plant.

As used herein, “abiotic stress” refers to negative impact of non-livingfactors on living organisms within the specific environment of theorganism. Exemplary abiotic stress conditions include, but are notlimited to drought, high heat, high salt, bright light, ultravioletlight, too high and too low temperatures, freezing, heavy metals andhypoxia. Abiotic stresses like salinity, drought or heat negativelyaffect plant growth and yield and belong to the most limiting factors ofagriculture worldwide (Wang et al., Planta, 218:1-14 (2003); Zurbriggenet al., Biotechnol Genet Eng Rev, 27:33-56 (2010)). For example,salinity, known to affect almost one fourth of arable land globally, isa two-phase stress composed of a rapid osmotic stress and a slower toxicone, resulting from Na⁺ ions accumulation and loss of K⁺ inphotosynthetic tissues (Negrao et al., Ann Bot, 119:1-11 (2017)). Saltstress reduces the rate of photosynthesis, leading to a decrease ofplant growth and crop yield (Hanin et al., Front Plant Sci., 7:1-17(2016)). However, in the context of global climate change and anincreasing world population, abiotic stress tolerant crops andsustainable solutions in agriculture are urgently needed to respond togrowing food demands (Coleman-Den, et al., Front Microbiol., 5:283(2014)).

The effect of salinity on plants includes two components: an osmoticcomponent, being the consequence of an altered osmotic pressure due toan increased salt concentration, and a toxic ion effect as a result ofthe high Na⁺ concentration in the shoot (Munns, et al., Annu Rev PlantBiol, 59:651-681 (2008); Shabala, et al., Ann Bot, 112:1209-1221(2013)). The toxic effects of Na⁺ accumulation result in prematuresenescence, leading to a decrease in photosynthesis efficiency andimpaired metabolic processes. Na⁺ also competes with K⁺ in membranetransport and enzymatic functions, reducing plant growth. Most plantcells possess mechanisms to counteract the harmful effects of Na⁺accumulation by retaining K⁺ and actively excluding Na⁺ in roots and/orsequestering Na⁺ in vacuoles in shoots (Munns, et al., Annu Rev PlantBiol, 59:651-681 (2008); Shabala, et al., Ann Bot, 112:1209-1221 (2013);Deinlein, et al., Trends Plant Sci, 19:371-379 (2014); Sun et al., PLoSOne, 10:e0124032 (2015)).

Arid regions cover about one quarter of the Earth's land surface andencompass many of the challenges for increasing agriculturalproductivity (Ezcurra, UNEP/Earthprint (2012)). In contrast to betterknown dryland farming, desert agriculture can function only when cropplants are under irrigation—usually with underground water with variouslevels of salinity (Cresswell, et al., Echo (1998)). Those areas faceextreme environmental conditions, characterized by high levels ofradiation, low rainfall, extreme temperatures, coarse soil which retainsvery little moisture, as well as low nutrients and typically highsalinity, which all strongly limit the yield of crops (Rewald, et al.,Wallingford: CABI, 196-218 (2012)). Although deserts appear to be hardlyinhabitable, a wide diversity of organisms has adapted to these extremeconditions. Plants along with their interacting microbial partners haveevolved sophisticated mechanisms such as production of osmoprotectants,Reactive Oxygen Species scavengers or Late Embryogenesis Abundantproteins to monitor the environment and reprogram their metabolism anddevelopment (Chaves, et al., Ann Bot, 89:907-916 (2002); Lebre, et al.,Nat Rev Microbiol, 15:285-296 (2017)). Therefore, this particularenvironment is an ideal reservoir to isolate and identify beneficialbacteria enhancing plant tolerance towards environmental stresses suchas drought, heat or salinity (de Zelicourt, et al., Mol Plant, 6:242-245(2013)).

As used herein, “plant growth promoting bacteria (PGPB)” refers tobeneficial bacteria that promote plant growth. PGPB can establishsymbiotic associations with plants and promote plant growth underoptimal growth conditions or in response to biotic and abiotic stresses(Obledo, et al., Plant Cell Tissue Organ Cult, 74:237-241 (2003);Marasco, et al., PLoS One, 7:e48479 (2012); Kaplan, et al., Am J Bot,100:1713-1725 (2013); Mengual, et al., J Environ Manage, 134:1-7 (2014);Cherif, et al., Environ Microbiol Rep, 7:668-678 (2015); Pieterse, etal., Annu Rev Phytopathol, 52:347-375 (2014)). Direct plantgrowth-promotion mechanisms include the acquisition of nutrients bynitrogen fixation, phosphate and zinc solubilization, or siderophoreproduction for sequestering iron. The modulation of phytohormone levels,such as auxin, ethylene, cytokinin or gibberellin, also largelycontributes to the beneficial properties of PGPB (Persello-Cartieaux, etal., Plant, Cell, Environ, 26:189-199 (2003); Vessey, Plant Soil,255:571-586 (2003); Hardoim, et al., Trends Microbiol, 16:463-471(2008)). Indirect mechanisms comprise the production of antimicrobialagents against plant pathogenic bacteria or fungi, or inducing systemicresistance against soil-borne pathogens (Pieterse, et al., Annu RevPhytopathol, 52:347-375 (2014); Glick, et al., Scientifica, 2012:1-15(2012)).

Several studies have shown that inoculation of commercial crops, such asmaize, strawberry and wheat by PGPBs under salt stress results in adecrease of Na⁺ and an increase of K⁺ in their shoots and leaves(Nadeem, et al., Microbiology, 25:78-84 (2006); Karlidag, et al., HorticSci, 48:563-567 (2013); Singh and Jha, Acta Physiol Plant, 38:110(2016)). The inoculation of A. thaliana and Trifolium repens (whiteclover) by Bacillus subtilis GB03 induced a decrease in the Na⁺ contentin shoots in both species accompanied by an increase or no change in theK⁺ content (Zhang, et al., Mol Plant-Microbe Interact, 21:737-744(2008); Han, et al., Front Plant Sci, 5:525 (2014)).

As used herein, “Arabidopsis” refers to the small flowering plantArabidopsis thaliana. Arabidopsis is used as a model system for studyingplant sciences, including genetics and plant development.

As used herein, “2-keto-4-methylthiobutyric acid (KMBA)” is anintermediate in the methionine salvage pathway. Oxidation of KMBA inceratin bacteria leads to the production of ethylene. KMBA can bespontaneously converted to ethylene by photo-oxidation or through theaction of plant peroxidases (Chague, et al., FEMS Microbiol Ecol,40:143-149 (2002)), which are abundantly present in the plant apoplast(Minibayeva, et al., Phytochemistry, 112:122-129 (2015); Karkonen, etal., Phytochemistry, 112:22-32 (2014)).

The term “plant substrate” refers to a substrate commonly used forgrowing plants, including plant seeds, plant roots and plant seedlings.Non-limiting examples of such plant substrates include, but are notlimited to soil, peat, compost, vermiculite, perlite, sand, clay, andcombinations thereof.

As used herein, “sodic soil” refers to soil with a disproportionallyhigh sodium content relative to other salts. Sodic soils arecharacterized by poor soil structure and low infiltration rate.Additionally, they are poorly aerated and difficult to cultivate.

II. Methods for Increasing Plant Tolerance to Abiotic Stresses

Methods and compositions for increasing tolerance to abiotic stresses inplants are provided. The methods include providing the plant with2-keto-4-methylthiobutyric acid (KMBA) exogenously (“exogenous KMBAmethod), alone or in combination with modifying the plant with amicrobial system that results in production of KMBA in the plant(“endogenous KMBA method”). The exogenous KMBA method include treatingseeds, seedlings or mature plants with 2-keto-4-methylthiobutyric acid(KMBA).

One embodiment provides a method for increasing salt tolerance in aplant by treating the plant with an amount of 2-keto-4-methylthiobutyricacid (KMBA) effective to increase salt tolerance in the plant relativeto an untreated plant (exogenous KMBA).

The endgenous KMBA method providing seeds, seedlings or mature plantswith a microbial system that results in production of KMBA in matureplants. In some embodiments, seeds or plant roots can be inoculated withor coated with a microbial system that results in production of KMBA inmature plants. A preferred microbial system Enterobacter sp. SA187. Inother embodiments, seeds, seedling and plants can be grown usingsubstrates containing an effective amount of Enterobacter sp. SA187 toinhibit or reduce abiotic stress in the plant. Suitable plant substratesinclude, but are not limited to soil, peat, compost, vermiculite,perlite, sand, clay and combinations thereof. Some embodiments providesa bacterium genetically engineered to express KMBA or increased levelsof KMBA relative to an unmodified bacterium, which can be used as amicrobial system that results in production of KMBA in mature plants. Ina preferred embodiment the bacterium is an endophytic bacterium. Theengineered bacterium can be used to inoculate plants and increasetolerance in the plant to abiotic stress conditions.

In another embodiment, Enterobacter sp. SA187 produces2-keto-4-methylthiobutyric acid (KMBA) in the plant (endogenous KMBA) topromote plant tolerance to salt stress.

Still another embodiment includes a combination of endogenous andexogenous KMBA treatment.

In some embodiments, the methods include providing the plant with one ormore additional plant-growth promoting bacteria and/or one or more plantgrowth-promoting rhizobacteria.

A. Exogenous 2-keto-4-methylthiobutyric acid (KMBA) Method

Methods for increasing salt tolerance in a plant by treating the plantwith an amount of KMBA effective to increase salt tolerance in the plantare provided. In one embodiment, KMBA can be delivered to mature plants.In other embodiments, KMBA can be used to irrigate seeds or seedlings.The plant can be treated with KMBA or KMBA in a solution.

In some embodiments, the amount of KMBA used to treat a plant is at aconcentration of 1.0 to 250 nM. The concentration of KMBA can be 1, 5,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, or 250nM. In a preferred embodiment, KMBA is present at a concentration of atleast 100 nM.

One embodiment provides a method for increasing plant tolerance toabiotic stress by treating the plant with a composition containing aneffective amount of KMBA. Exemplary abiotic stress conditions include,but are not limited to drought, high heat, high salt, bright light,ultraviolet light, too high and too low temperatures, freezing, heavymetals and hypoxia. In one embodiment, the abiotic stress can be watersalinity, soil salinity, or both.

In one embodiment, plants are treated with an amount of KMBA effectiveto increase salt tolerance in the plant. The KMBA can be delivered tothe plant in solution. In one embodiment, KMBA is delivered in anaqueous solution. The aqueous solution can be water, salt water, orseawater.

Some embodiments provide a method of delivering KMBA to plants in asolution. The solution can contain 1-10% salt. In some embodiments thesolution contains 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%,6.5%, 7%, 7.5%, 8%, 8.5%, 9%, or 9.5% salt. In a preferred embodiment,the solution contains 3.5% salt. The salt can be selected from the groupcontaining salts of sodium, chloride, sulfate, magnesium, calcium,potassium, or combinations thereof.

B. Edogenous 2-keto-4-methylthiobutyric acid (KMBA) Method

Enterobacter sp. SA187 is a bacterium that was previously isolated fromthe desert pioneer plant Indigofera argentea Burm.f. (Fabaceae) (Lafi,et al., Genome Announc, 9-10 (2017); Andres-Barrao, et al., FrontMicrobiol, 8:1-21 (2017)). SA187 was found to colonize both surface andinner plant root and shoot tissues and to modify several plantphytohormone pathways. Enterobacter sp. SA187 was shown to significantlyincrease the yield of the agronomically important crop alfalfa (Medicagosativa) in field trials under both normal and salt stress conditions,demonstrating that SA187 has a high potential to improve agricultureunder desert conditions. Gene expression analysis of SA187-containingplants indicated an upregulation of the methionine salvage pathway uponplant colonization, increasing the production of2-keto-4-methylthiobutyric acid (KMBA), which is known to be convertedinto ethylene in planta.

Methods for providing plants with tolerance to abiotic stress conditionsare disclosed in WO 2017/125894. The methods include inoculating theseed or plant with an effective amount (10⁶-10⁸ bacteria/ml) of SA187 toprovide the seed or plant with resistance to the abiotic stressconditions. The inoculation of the plant can be in the rhizosphere ofthe plant. The rhizosphere is the area around a plant root that isinhabited by a unique population of microorganisms. Alternatively, theplant root can be inoculated directly. In certain embodiments, the plantroot is coated with SA187. The SA187 is preferably the bacterium whosegenome contains SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, ora combination thereof. More preferably, the SA187 genome contains all ofSEQ ID NO:1-13.

Another embodiment use a seed coating composition containing SA187. TheSA187 can be encapsulated with a non-toxic, biodegradable coating. Theseed coating composition can also contain a coating adhesive. Exemplaryseed coating compositions contain gelatin, cellulose, alginate, xanthum,or a combination thereof. Certain seed coating compositions can havemultiple layers.

Other embodiment provides a method of improving growth of a seed orplant under abiotic stress conditions by growing the seed or plant in aplant substrate, wherein the plant substrate includes an effectiveamount of SA187 to colonize the seed or a root of the plant to provideabiotic stress resistance to the seed or plant.

One embodiment provides a plant substrate containing an effective amountof a species of the genus Enterobacter to inhibit or reduce abioticstress in the plant. In a preferred embodiment, the Enterobacter speciesis SA187. Suitable plant substrates include, but are not limited tosoil, peat, compost, vermiculite, perlite, sand, clay and combinationsthereof. Typically, the plant substrates contain 10⁶ to 10⁹ bacteriaper/g of substrate.

The disclosed methods can use bacteria genetically engineered to expressKMBA or increased levels of KMBA relative to an unmodified bacterium,which can be used as a microbial system that results in production ofKMBA in mature plants. Bacteria can be genetically engineered tooverexpress KMBA or increased levels of KMBA relative to an unmodifiedbacterium using methods known in the art. The data in this applicationshows that the expression level of most of the genes encoding proteinsinvolved in the methionine salvage pathway were upregulated in SA187upon plant colonization compared with bacteria incubated for 4 h inliquid ½ MS with or without 100 mM NaCl in the absence of plants. Thus,the disclosed methods include engineering bacteria to overexpress one ormore genes involved in the methionine salvage pathway.

Many organisms including bacteria utilize the methionine salvage (FIG.7F) pathway recycling methylthioadenosine (MTA) to methionine. In afirst step in E. coli, MTA is hydrolysed by a nucleosidase (EC 3.2.2.16,coded by gene pfs, now mtnN, for methylthioadenosine nucleosidase).Additional genes include: tyrB encoding a wide spectrum aminotransferasethe gene encoding MtnA (methylthioribose-1-phosphate isomerase), mtnA,mtnB, which codes for a methylthioribulose-1-phosphate dehydratase; anenolase-phosphatase, encoded by gene mtnC; mtnD (ykrZ) , which codes forthe aci-reductone dioxygenase (Sekowska. et al. BMC Microbiology 2004,4:9; mtnK was identified encoding methylthioribose kinase (Sekowska, etal., BMC Microbiology 2001, 1:15); metK, encoding S-adenosylmethionine(SAM) synthetase; speE encoding spermidine synthetase; speD, encodingS-adenosylmethionine decarboxylase (Gao, et al., MBio, 5(2)c0079-14,(2014); Sekowska, et al., J. Mol. Microbiol. Biotechnol. (2000) 2(2):145-177)). Additionally, the bacteria can be engineered to expressL-amino acid deaminase (L-AAD) from Proteus vulgaris as disclosed forexample in Hossain, et al., PLOS One, 9(12):e114291 (2014), for one-stepbiosynthesis of α-keto-γ-methylthio butyric acid from E. coli fromL-methionine. Preferred bacteria include SA187 and E. coli. However, asuitable bacterium can be selected from the list of bacteria suitablefor co-inoculation, described below.

Seed Coating Compositions

One embodiment provides a seed coating composition having at least onelayer coating all or part of the seed, wherein at least one layercontains an effective amount of KMBA and/or SA187 to provide the seed orthe plant that grows from the seed with resistance to abiotic stressconditions. The SA187 can be from 10⁶/ml to 10⁹/ml of seed coatingcomposition.

Another embodiment provides a seed coating composition containing KMBA.The KMBA can be encapsulated with a non-toxic, biodegradable coating.The seed coating composition can also contain a coating adhesive.Exemplary seed coating compositions contain gelatin, cellulose,alginate, xanthum, or a combination thereof. Certain seed coatingcompositions can have multiple layers.

In another embodiment, the seed coating composition contains multiplelayers for example, 2, 3, 4 or 5 or more layers. KMBA, SA187 and/or oneor more additional plant growth promoting bacteria can be in any or allof the layers of the seed coating composition; however, at least one ofthe layers of a multiple layer seed coating composition contains aneffective amount of KMBA and/or SA187 to provide the seed or the plantgrowing from the seed with resistance to abiotic stress conditions.Preferably, the KMBA and/or SA187 or other plant growth promotingbacteria are in the layer adjacent to the surface of the seed.

In one embodiment, at least one layer contains guar gum, derivativeguar, polyacrylamide, poly(methacrylic acid), poly(acrylic acid),polyacrylate, poly(ethylene glycol), phosphonate-end capped polymers,polyethyleneoxide, poly(vinyl alcohol), polyglycerol,polytetrahydrofuran, polyamide, hydroxypropyl guar, carboxymethyl guar,carboxymethyl hydroxypropyl guar, starch, derivatized (e.g., cationic)starch, corn starch, wheat starch, rice starch, potato starch, tapioca,waxy maize, sorghum, waxy sarghum, sago, dextrin, chitin, chitosan,alginate compositions, xanthan gum, carageenan gum, gum karaya, gumarabic, pectin, cellulose, hydroxycellulose, hydroxyalkyl cellulose,hydroxyethyl cellulose, carboxymethylhydroxyethyl cellulose,hydroxypropyl cellulose, a derivative of any of the foregoing or acombination of any of the foregoing. As non-limiting examples, the layercan contain a 90 wt % derivatized guar and 10 wt % starch (orderivatized starch) mixture, or a 60 wt % hydroxypropyl guar and 40 wt %carboxymethyl hydroxypropyl guar mixture.

In some embodiments, the layer can act as a carrier coating. Fungicidesand beneficial microbes that protect the seed and emerging seedling arecarried in the carrier coating. For example, alfalfa seed coating withincorporated KMBA and/or SA187 is used to inoculate the field in whichdesired crop plants are planted or are growing.

Another embodiment provides agglomerates of seed. The agglomerate orgrouping of seed is a grouping of 2 or more individual seeds together.The seeds can be for the same plant or for different plants. In anotherembodiment, the agglomerate is a grouping of more than 5 individualseeds together. In a further embodiment, the agglomerate is a groupingof more than 10 individual seeds together. In yet another embodiment,the agglomerate is a grouping of more than 25 individual seeds together.In yet a further embodiment, the agglomerate is a grouping of more than50 individual seeds together. In another embodiment, the agglomerate isa grouping of more than 100 individual seeds together.

1. Seed Agglomerates

The agglomeration of seed can aid in the application of the seed coatingcomposition because the seed coating composition, when using anagglomeration of seed, can be shaped or formed to be consistent in shapeor form. For example, the agglomeration can be formed as spherical orsubstantially spherical, thus allowing the seed coating composition tobe likewise substantially spherical. This can allow for improved or moreconsistent casting or spraying, can minimize the occurrence of blockageor clogging of the nozzles, hoses, etc. due to uneven particle sizedistribution. Typically, a binder or adhesive is utilized to bunch(e.g., agglomerize) the grouping of seeds together.

The agglomeration can also aid in seed or seedling establishment as alayer of the wetting agent (or other layer than affects the soil) can beconcentrated to a local area of soil, thus, increasing its chance ofwetting the soil surrounding the seed(s). the agglomeration can alsopromote survival by allowing the seeds, when germinating into seedlings,to generate sufficient force to penetrate hydrophobic areas or soil suchas, for example, a hydrophobic (i.e., encrusted) soil surface

In one embodiment, the seed coating composition contains anagglomeration of seeds of from between 2 seeds to 100 seeds, typicallybetween 2 to 50 seeds, typically between 2 to 25 seeds; and at least onelayer selected from the group consisting a layer of a filler, a layer ofa binding agent, a layer of a wetting agent, a layer of an anti-bacteriaagent, a layer of an active ingredient and any combination thereof.

In one embodiment, the seed coating composition is of substantiallyuniform size of from between 10 micrometers and 4 mm in diameter. Inanother embodiment, the seed coating composition is of substantiallyuniform size of from between 25 micrometers and 2 mm in diameter. In afurther, the seed coating composition is of substantially uniform sizeof from between 500 micrometers and 2 mm in diameter.

2. Binder

The seed coating composition can also contain a binder as one of thelayers, the binder is sometimes referred to as an adhesive. In oneembodiment, the binder can include but is not limited to molasses,granulated sugar, alginates, karaya gum, guar gum, tragacanth gum,polysaccharide gum, mucilage or any combination of the foregoing. Inanother embodiment, the binder is chosen from, but is not limited to,gelatin, polyvinyl acetates, polyvinyl acetate copolymers, polyvinylalcohols, polyvinyl alcohol copolymers, celluloses (includingethylcelluloses and methylcelluloses, hydroxypropylcelluloses,hydroxymethyl celluloses, hydroxymethylpropyl-celluloses),polyvinylpyrolidones, dextrins, malto-dextrins, polysaccharides, fats,oils, proteins, gum arabics, shellacs, vinylidene chloride, vinylidenechloride copolymers, calcium lignosulfonates, acrylic copolymers,starches, polyvinylacrylates, zeins, carboxymethylcellulose, chitosan,polyethylene oxide, acrylimide polymers and copolymers, polyhydroxyethylacrylate, methylacrylimide monomers, alginate, ethylcellulose,polychloroprene, syrups or any combination of the foregoing.

3. Active Ingredients

The seed coating compositions can also include one or more activeingredients in one or more of the layers of the coating. Compoundssuitable as active ingredients, which in some embodiments form all orpart of at least one layer of the seed coating composition, include butare not limited to herbicides, plant growth regulators, crop desiccants,fungicides, insecticides, insect repellants, and combinations thereof.Suitable pesticides include, for example, triazine herbicides;sulfonylurea herbicides; uracils; urea herbicides; acetanilideherbicides; and organophosphonate herbicides such as glyphosate saltsand esters. Suitable fungicides include, for example, nitrilo oximefungicides; imidazole fungicides; triazole fungicides; sulfenamidefungicides; dithio-carbamate fungicides; chloronated aromatic; anddichloro aniline fungicides. Suitable insecticides, include, forexample, carbamate insecticides; organo thiophosphate insecticides; andperchlorinated organic insecticides such as methoxychlor. Suitablemiticides include, for example, propynyl sulfite; triazapentadienemiticides; chlorinated aromatic miticides such as tetradifan; anddinitrophenol miticides such as binapacryl. Other active ingredients caninclude adjuvants, surfactants, and fertilizers.

4. Filler

The seed coating composition can also include at least one filler as allor part of a layer. In one embodiment, the filler is selected from thegroup consisting of wood flours, clays, activated carbon, carbohydrates,sugars, dextrins, maltodextrins, diatomaceous earth, cereal flours,wheat flour, oat flour, barley flour, fine-grain inorganic solids,calcium carbonate, calcium bentonite, kaolin, china clay, talc, perlite,mica, vermiculite, silicas, quartz powder, montmorillonite or mixturesthereof.

5. Nutrients

The seed coating composition can also contain a nutrient such as amicronutrient or macronutrient in one or more layers of the seed coatingcomposition. The nutrient can be in all or part of a layer. The nutrientcan also be included with the grouping of seeds as part of the binder oradhesive. “Nutrient” as used herein can refer to an additive orsubstance utilized by plants, grasses, shrubs for plant, grass, andshrub growth, respectively. Macronutrients can be utilized in largeramounts by plants, grasses, etc. in proportionally larger amountsrelative to micronutrients. Nutrients include but are not limited tomanganese, boron, copper, iron, chlorine, molybdenum, and zinc,potassium, nitrogen, calcium, magnesium phosphorus and sulfur, amongothers. The seed coating compositions can include various combinationsand relative amounts of individual macronutrients.

Co-Inoculation

Plants and seeds can be co-inoculated with SA187 and one or more otherplant growth-promoting bacteria or rhizobacteria to provide the seeds orplants with resistance or tolerance to abiotic stress conditions.Co-inoculation is based on mixed inoculants, combination ofmicroorganisms that interact synergistically, or when microorganismssuch as Azospirillum are functioning as “helper” bacteria to enhance theperformance of other beneficial microorganisms. In the rhizosphere thesynergism between various bacterial genera such as Bacillus, Pseudomonasand Rhizobium has been demonstrated to promote plant growth anddevelopment. Compared to single inoculation, co-inoculation can improvethe absorption of nitrogen, phosphorus and mineral nutrients by plants.

Suitable bacteria that can be co-inoculated with SA187 include but arenot limited to Pseudomonas putida, Pseudomonas aeruginosa, Klebsiellasp., Enterobacter asburiae, Rhizobium sp. (pea), Mesorhizobium sp.,Acinetobacter spp., Rhizobium sp.(lentil), Pseudomonas sp. A3R3,Psychrobacter sp. SRS8, Bradyrhizobium sp., Pseudomonas aeruginosa 4EA,Pseudomonas sp., Ochrobactrum cytisi, Bacillus species PSB10,Paenibacillus polymyxa, Rhizobium phaseoli, Rahnella aquatilis,Pseudomonas fluorescens, Ralstonia metallidurans, Azospirillumamazonense, Serratia marcescens, Enterobacter sp., Burkholderia,Pseudomonas jessenii, Azotobacter sp., Mesorhizobium ciceri, Azotobacterchroococcum, Klebsiella oxytoca, Pseudomonas chlororaphis, Baciilussubtilis, Gluconacetobacter, diazotrophicus, Brevibacillus spp.,Bravibacterium sp., Xanthomonas sp. RJ3, Azomonas sp. RJ4, Pseudomonassp. RJ10, Bacillus sp. RJ31, Bradyrhizobium japonicum, Variovoraxparadoxus, Rhodococcus sp., Flavobacterium, Sphingomonas sp,Mycobacterium sp, Rhodococcus sp, Cellulomonas sp., Azospirillum sp.,Azospirillum brasilense, Rhizobium meliloti, Kluyvera ascorbata,Rhizobium cicero, Rhizobium leguminosarum, Paenibacillus polymyxa strainA26, a Alcaligenes faecalis strain AF, and combinations thereof.

In some embodiments, the inoculation method does not include inoculatingthe plant or seeds with SA187. In these embodiments, the exogenous KMBAmethod is employed in seeds/plants inoculated one or more other plantgrowth-promoting bacteria or rhizobacteria to provide the seeds orplants with resistance or tolerance to abiotic stress conditions

C. Coating Techniques

Equipment utilized to for coating seeds with the disclosed seed coatingcompositions include, but are not limited to drum coaters, rotarycoaters, tumbling drums, fluidized beds and spouted beds to name a few.The seeds can be coated via a batch or continuous coating process.

The seeds can be separated prior to coating which, in one embodiment,utilizes mechanical means such as a sieve. The separated seeds can thenbe introduced into a coating machine having a seed reservoir. In oneembodiment, the seeds in the mixing bowl are combined with one or moreof the coatings described herein and adhered with a binder or adhesive.

In one embodiment of the process, one or more layers as described hereincan be added to coat the seed or agglomeration. Outer layers can beintroduced sequentially to the rotating drum.

In another embodiment, agglomerators or agglomerator devices may also beutilized. Coating is performed within a rotary coater by placing seedswithin a rotating chamber, which pushes the seeds against the insidewall of the chamber. Centrifugal forces and mixing bars placed insidethe coater allow the seed to rotate and mix with a coating layer. Binderor other coating materials can be pumped into the proximate center ofthe coater onto an atomizer disk that rotates along with the coatingchamber. Upon hitting the atomizer disk, liquid adhesive is thendirected outward in small drops onto the seed.

In one embodiment, seed coating techniques include, for example, seed ina rotating pan or drum. Seed is then misted with water or other liquidand then gradually a fine inert powder, e.g., Diatomaceous earth, isadded to the coating pan. Each misted seed becomes the center of a massof powder, layers, or coatings that gradually increases in size. Themass is then rounded and smoothed by the tumbling action in the pan,similar to pebbles on the beach. The coating layers are compacted bycompression from the weight of material in the pan. Binders often areincorporated near the end of the coating process to harden the outerlayer of the mass. Binders can also reduce the amount of dust producedby the finished product in handling, shipping and sowing. Screeningtechniques, such as frequent hand screening, are often times utilized toeliminate blanks or doubles, and to ensure uniform size. For example,tolerance for seed coating compositions described herein can be about1/64th inch (0.4 mm), which is the US seed trade standard for sizing.

In yet another embodiment, the seed coating compositions and methodsdescribed herein comprises “in situ coating”. In situ coating means, inone embodiment, where a raw or non-coated seed is implanted in a hole,cavity or hollowed area in the ground and immediately or soon thereaftera coating composition is sprayed or applied directly into the hole,cavity or hollowed area to surround or partially surround the seed.Typically, the application of the seed as well as application of thecoating composition are performed mechanically, but is understood thateither or both of the referenced applications can be performed manuallyas well.

The coating can also be applied to a seed by spraying, dipping orbrushing.

D. Cultivation Methods

One embodiment provides a method of cultivating a plant by plantingseeds or seedlings of the plant and irrigating the seeds or seedlingswith a saline solution containing KMBA. The plant can be a forage crop.In one embodiment the forage crop is selected from the group containingrice, wheat, sugarcane, maize, soybean, cotton, vegetables, rape,mustard, sorghum, millet, grass, Brassica spp., rapeseed, barley, hay,and alfalfa.

Another embodiment provides a method of cultivating plants in sodic soilby contacting the plant in the sodic soil with an effective amount ofKMBA to increase salt tolerance in the plant.

In embodiments combining the exogenous and endogenous KMBA methods,seeds or plants inoculated with an effective amount of SA187 to providethe seed or plant with resistance or tolerance to abiotic stresscondition are cultivated in sodic soil by contacting the plant in thesodic soil with an effective amount of KMBA to increase salt tolerancein the plant. Preferred seeds and plants, preferably roots that can beinoculated with or coated with SA187 include, but are not limited toalfalfa, cotton, wheat, maize, soybean, oat, barley, potato, and sugarbeets.

E. Irrigation Solution

An irrigation solution to increase salt tolerance in plants is provided.

The irrigation solution contains 1.0 to 250 nM KMBA in an aqueous salinesolution. In one embodiment the solution contains 1, 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, or 250 nM of KMBA. Ina preferred embodiment, KMBA is present at a concentration of at least100 nM.

In one embodiment the aqueous saline solution is seawater. The aqueoussolution can contain 1% to 10% salt. In some embodiments the solutioncontains 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%,7.5%, 8%, 8.5%, 9%, or 9.5% salt. In a preferred embodiment, thesolution contains 3.5% salt.

EXAMPLES Data Submission

RNAseq data are available under the ID GSE102950 which is incorporatedby reference in its entirety.

The isolation of SA0187 is previously disclosed in WO 2017/125894.Briefely, Enterobacter sp. SA187 (endophytic bacteria) was isolated fromsurface sterilized root nodules formed on roots of pioneer plantIndigofera argentea Burm.f. (Fabaceae). Plants were collected fromdifferent regions in the Jizan area (16° 56.475′ N, 42° 36.694′ E) ofSaudi Arabia. SA187 has been shown to possess plant growth promotingactivities, such as the production of siderophores and indole aceticacid (IAA).

Sequencing

The genomic DNA of SA187 was extracted using the Qiagen's DNeasy bloodand tissue kit following the manufacturer protocol. The DNA was thensequenced using paired-end Illumina MiSeq and the library preparationwas constructed as described previously (1). Contig assembly was donewith Spades assembler version 3.6 (4) with a 1 KB contig 52 cutoff size.

Total RNA was extracted from 5-day-old plants inoculated or not withSA187 and transferred for 10 more days on ¹/₂ MS plates with or without100 mM NaCl using the Nucleospin RNA plant kit (Macherey-Nagel),including DNase treatment, and following manufacturer's recommendations.

RNA samples were used for Illumina HiSeq deep sequencing (Illumina HiSeq2000, Illumina). Three biological replicates were processed for eachsample. Paired-end sequencing of RNA-seq samples was performed usingIllumina GAIIx with a read length of 100 bp. Reads werequality-controlled using FASTQC(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trimmomaticwas used for quality trimming⁸. Parameters for read quality filteringwere set as follows: Minimum length of 36 bp; Mean Phred quality scoregreater than 30; Leading and trailing bases removal with base qualitybelow 3; Sliding window of 4:15. TopHat v2.0.9⁹ was utilized foralignment of short reads to the Arabidopsis thaliana genome TAIR10(Supplementary Table 1), Cufflinks v2.2.0¹⁰ for transcript assembly anddifferential expression. To identify differentially expressed genes,specific parameters (p-value: 0.05; statistical correction: BenjaminiHochberg; FDR: 0.05) in cuffdiff were used. Post-processing andvisualization of differential expression were done using cummeRbundv2.0.0¹¹. Gene is considered as regulated if fold change>1log2^(|0.6|)and q-value<0.05. Results were confirmed by analyzing 12 genes usingqPCR method.

For qPCR analysis, plant RNAs were purified as described previously. Forbacterium, SA187 incubated 4 h in ½ MS or ½ MS+100 mM, at 28° C., underthe dark, were used for RNA extraction, using the RiboPure™ RNAPurification Kit, bacteria (Ambion) following manual instructions forGram-(−) bacteria, with the exception that no beads were used duringbacterial lysis. RNA extraction was followed by DNAseI treatment inorder to obtain purified total RNA.

cDNAs were synthetized using SuperscriptIII (Invitrogen), 1 μg of totalRNA and oligo-dT following manufacturer's recommendations. ForArabidopsis gnee expression analyses, ACTIN2 (At3 g18780) andUBIQUITIN10 (At4 g05320) were used as reference genes. For SA187 geneexpression analyses, infB, rpoB and gyrB were used as reference genes.All reactions were done in a CFX96 Touch™ Real-Time PCR Detection System(BIO-RAD) as follows: 50° C. for 2 min, 95° C. for 10 min; 40×[95° C.for 10 sec and 60° C. for 40 sec]; and a dissociation step to validatethe PCR products. All reactions were performed in three biologicalreplicates, and each reaction as a technical triplicate. Gene expressionlevels were calculated using Bio-Rad CFX manager software.

Hierarchical Clustering and Gene Family Enrichment

Arabidopsis regulated genes were used to generate HCL tree using MultiExperiment Viewer (MeV 4.9.0 version, TM4,https://sourceforge.net/projects/mev-tm4/files/mev-tm4/MeV %204.9.0/).Raw data were normalized for every gene and transformed in log2.Hierarchical clustering was performed using Euclidian distances, averagelinkage and leaf order optimization. Heat colors indicate log2 foldchange. Gene enrichment analyses were performed using AmiGO website(http://amigol.geneontology.org/cgi-bin/amigo/term_enrichment). Eachcluster were analyzed using default parameter.

Results

De novo assembly of MiSeq reads for Enterobacter sp. SA187 resulted in14 contigs with a total length of 4,404,403 bp and a mean contig size of314,600 bp. The N50 was 2,296,004 bp and the L50 has been reached in 1contig. The GC content of this draft genome was 56%. Megablastcomparison of the SA187 concatenated contigs against the NCBI referencegenome database (http://www.ncbi.nlm.nih.gov/genome/) revealed the 57closest relative genomes being Enterobacter sacchari SP1 with a coverageof 63% and sequence identity of 95% (Accession number NZ_CP007215.2).The annotation of Enterobacter sp. SA187 was carried out using thedefault INDIGO pipeline (7) with the exception of open reading frame(ORFs) prediction by FragGeneScan. The annotation of SA187 resulted in3,087 ORFs, 9 rRNA, 75 tRNA, and 145 ncRNA.

The annotation predicted a number of siderophore pathway genes such asentE, entC, entA, entB, entF, as well as entS, an MFS transporter ofenterobactin. An ABC transporter involved in iron uptake, sitABCD, wasalso found, as well as five copies of the iron complex outer membranereceptor (fhuA) and a TonB-dependent outer membraneiron-enterobactin/colicin (fepA). Generally, PGPR bacteria enhance plantgrowth through the synthesis of IAA from tryptophan via indole pyruvateas the main pathway (9). The SA187 genome harbors a number of genesinvolved in this pathway, but lacks the gene encoding for indolepyruvatedecarboxylase (ipdC). Moreover, the SA187 genome codes for the enzymetryptophanase (TnaA) (EC: 4.1.99.1), which can transform tryptophaneinto indole.

Genome of Enterobacter sp. SA187 has been deposited at DBJ/EMBL/GenBankunder accession number MORB00000000 (which is incorporated herein in itsentirety. The genome of SA187 contains SEQ ID NO:1-13.

Results

Based on 16S rDNA sequencing and comparison, SA187 was suggested tobelong to the Enterobacter genus, with high homology with Enterobacterkobei strains.

The 16S ribosomal RNA gene sequence (or 16S rRNA) is deposited atDDBJ/EMBL/GenBank under the accession no KY194757.

Based on the 16S rRNA gene sequence the SA187 is closely related toEnterobacter kobei CCUG 49023^(T) and Enterobacter aerogenes strain KCTC2190 with 99% sequence similarity. SA187 comprises SEQ ID NO:1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, ora combination thereof.

Example 1 Enterobacter sp. SA187 increases Alfalfa Yield in Field Trialsunder Field Conditions

Methods

Field Trials

To inoculate alfalfa seeds (Medicago sativa var. CUF 101), a slurry wasprepared consisting of sterilized peat, a broth culture of SA187, andsterilized sugar solution (10%) in the ratio 5:4:1 (w/v/v).Subsequently, alfalfa seeds were coated with the slurry at a rate of 50mL·kg⁻¹. As a control, seeds were coated with a similar mixture withoutbacteria. Field trials were conducted at the experimental station inHada Al-Sham (N 21° 47′47.1″ E 39° 43′48.8″), Saudi Arabia, in winterseasons 2015-2016 and 2016-2017. The experiment was a randomizedcomplete block design with a split-split plot arrangement of fourreplicates in the season 2015-2016 season and three replicates in the2016-2017 season, plots (2×1.5 m) with seed spacing 20 cm row-to-row.The field was irrigated using ground water with two different salinitylevels: low salinity (EC=3.12 dS·m⁻¹), and high salinity (EC=7.81dS·m⁻¹). The soil had an average pH 7.74 and salinity EC=1.95 dS·m⁻¹.Agronomical data (plant height, fresh biomass, and dry biomass) wererecorded every 25-30 days from each harvest; three harvests were done inthe first season, four harvests in the second season.

Results

Since SA187 was an outstandingly performing bacterial isolate in aprevious screen using Arabidopsis as a model plant (Andres-Barrao, etal., Front Microbiol, 8:1-21 (2017)), the potential agronomic use ofSA187 as a biological solution for agriculture was evaluated. Therefore,the beneficial activity of SA187 on different growth parameters of thecrop plant alfalfa (Medicago sativa), which is largely used as animalfeed in different regions of the world, was tested. Alfalfa seeds werecoated with SA187 and tested in parallel with mock-coated seeds in theexperimental fields' station near the city of Jeddah, Saudi Arabia. Arandomized complete block design with a split-split plot arrangementwith different replicates was used over two subsequent growth seasons(2015-2016 and 2016-2017). In the first growing season, using low salinewater (EC=3.12 dS·m⁻¹) irrigation, SA187-inoculated alfalfa plantsexhibited an increase of 25% for plant height, leading to an increase ofboth fresh and dry biomass by 16% and 14%, respectively (FIG. 1A, B, C).Interestingly, using high saline irrigation water (EC=7.81 dS·m⁻¹), asimilar impact on the plant growth was observed. In the growing season2016-2017, although there was no clear difference in the alfalfa yieldbetween plots irrigated with low and high saline water, a beneficialimpact of SA187 was still observed on both experimental plots irrigatedwith low and high saline water (FIG. 1D, E, F). The lowered increase ingrowth parameters in the second season is most likely due to exceptionalrainfalls in that period. In conclusion, SA187 can efficiently improvecrop productivity under extreme agricultural conditions.

Example 2 Enterobacter sp. SA187 enhances salt tolerance in Arabidopsisthaliana

Methods

Endophytic Bacteria, Plant Material, Growth Condition and PhysiologicalExperiments

Enterobacter sp. SA187 was previously isolated from root nodules of theleguminous pioneer plant Indigofera argentea in the Jizan region ofSaudi Arabia (Lafi, et al., Genome Announc, 9-10 (2017); Andres-Barrao,et al., Front Microbiol, 8:1-21 (2017)). The complete genome sequence ofEnterobacter sp. SA187 is known in the art (Andres-Barrao, C., et al.,Front Microbiol. 8:1-21 (2017)). Arabidopsis seeds were obtained eitherfrom publicly available collections or provided by Dr. Jean Colcombet(IPS2, Orsay, France). The following mutant lines used in this studywere published previously: the JA-receptor coil-1 mutant (Xie, et al.,Science, 280:1091-1094 (1998)), JA-insensitive jar1-1 (Staswick, et al.,Proc Natl Acad Sci USA, 89 :6837-6840 (1992)), the ABA biosynthesisaba2-1 mutant (Schwartz, et al., Plant Physiol, 114 :161-166 (2997)),the ABA receptor quadruple pyr1-1pyl1-1pyl2-1pyl4-1 mutant (Park et al.,Science, 324:1068-1071 (2009)), the ethylene insensitive ein2-1 (Guzmán,et al., Plant Cell, 2:513-523 (1990)) and ein3-1 mutants (Roman, et al.,Genetics, 139:1391-1409 (1995)), the heptuple ethylene-biosynthesisdeficient mutant acs1-1acs2-1acs4-1acs5-2acs6-lacs7-lacs9-1 (Tsuchisaka,et al., Genetics, 183:979-1003 (2009)), and the ethylene-dependentpEBF2::GUS reporter (Konishi and Yanagisawa, Plant J, 55:821-831(2008)).

Prior to every experiment, A. thaliana seeds were surface sterilized 10min in 70% ethanol +0.05% sodium dodecyl sulfate on a shaker, washed 2times in 96% ethanol and let to dry. To ensure SA187-inoculation,sterilized seeds were sown on ½ MS plates (Murashige and Skoog basalsalts, Sigma) containing SA187 (2·10⁵ cfu·ml⁻¹), stratified for 2 daysat 4° C. in the dark and then placed vertically to growth conditions for5 days. The ½ MS plates with SA187 were prepared by addition of acalculated amount of bacterial suspension to pre-cooled agar mediumduring plate preparation.

Average length of root hairs was determined based on images of 5-day-oldroots (1 image per root at constant distance from the root tip, 25seedlings per condition) or 16-day-old roots (along the whole primaryroot length grown after transfer) captured by a Nikon AZ100M microscopeequipped with an AZ Plan Apo 2× objective and a DS-Ri1 camera (Nikon).All root hairs in focus were measured using ImageJ. Average values andstandard deviations were calculated from 10% longest root hairs toeliminate non-developed root hairs and describe the maximal elongationcapacity of root hairs.

For salt stress tolerance assays, 5-day-old seedlings were transferredonto ½ MS plates with or without 100 mM NaCl (Sigma). Primary rootlength was measured every 2 days using ImageJ software after scanningthe plates. Lateral root density was evaluated as detectable number oflateral roots under a stereo microscope divided by the primary rootlength. Fresh weight of shoots and roots was measured 12 days aftertransfer of seedlings. Dry weight was measured after drying shoota androots for 2 days at 70° C. Following Koch's postulate, SA187 wasre-isolated from Arabidopsis root system at the end of an initialexperiment to confirm the genotype of the inoculated strain. To addressthe ethylene involvement in Arabidopsis adaptation to salt stress, ACC(1-aminocyclopropane-1-carboxylic acid, Sigma), KMBA(2-keto-4-methylthiobutyric acid, Sigma), AVG (aminoethoxyvinylglycine,Sigma), AgNO3 (silver nitrate, Sigma) were added into pre-cooled ½ MSagar medium together with 100 mM NaCl. For DNPH(2,4-dinitrophenylhydrazine, Sigma), 5 mM solution was prepared bysolubilizing DNPH into 2M HCl as described previously (Primrose, J GenMicrobiol, 98:519-528 (1977)), then the solution was diluted untilreaching 1 mM, and equilibrated to the same pH as MS medium (pH 5.8)using 2M KOH. DNP was used at final concentration 3 μM.

All plants were grown in long day conditions in growth chambers(Percival; 16 h light/8 h dark, 22° C.). Each experiment was performedat least in three biological replicates.

Results

To better understand the molecular mechanism how SA187 confers stresstolerance to plants, model genetic plant A. thaliana was used. Thecapacity of SA187 to affect the early stages of Arabidopsis developmentunder normal conditions (½ MS agar medium, 22° C., 16 h of light) wasassessed. When compared to mock-inoculated plants, SA187 had noinfluence on the germination rate of Arabidopsis seeds (FIG. 2A), andapart from considerably longer root hairs (FIG. 2B, C), 5-day-oldseedlings showed no morphological changes. Similarly, after transferonto new ½ MS plates, no differences between 17-day-old mock- andSA187-inoculated seedlings were recorded, when measuring root length(FIG. 2D-E), lateral root density (FIG. 2J), shoot morphology (FIG. 2E),or root and shoot fresh and dry weight of seedlings (FIG. 2F-I)indicating that SA187 has no effect on Arabidopsis development undernormal growth conditions.

On the other hand, the stress tolerance promoting capacity of SA187 onArabidopsis growth was highlighted under salt stress. Five days aftergermination, SA187- and mock-inoculated seedlings were transferred onto½ MS agar plates supplemented with 100 mM NaCl, and the same growthparameters were evaluated as above up to 12 days after the transfer tosalt plates. SA187-inoculated plants showed stress tolerance promotingactivity on salt stress: the shoot and root systems of SA187-inoculatedplants were significantly more developed than those of mock-inoculatedplants (FIG. 2E-J). While primary root length was similar between SA187-and mock-inoculated plants (FIG. 2D), lateral root density wassignificantly increased (FIG. 2J). Similarly to 5-day-old seedlings,SA187-inoculated plants at this stage had more than twice longer roothairs compared to the mock-inoculated ones under both normal and saltstress conditions. Moreover, it was proven that the beneficial activityof SA187 was largely linked to living bacterial cells as dead SA187cells killed by heat-inactivation did not induced any beneficialactivity.

Overall, SA187 strongly enhanced Arabidopsis growth with increased totalfresh and dry weights of both shoot and root under salt stressconditions.

Example 3 Enterobacter sp. SA187 Modifies Root and Shoot K⁺ levels

Methods

Na⁺ and K⁺ Content Determination

Dry rosettes and root systems were weighted. All samples were measuredindividually except for salt-treated root systems, whereby pools ofthree root systems were measured to ensure proper weight measurements.Sodium and potassium concentrations were prepared for shoot and root drysamples by adding 1 mL of freshly prepared 1% HNO₃ (nitric acid, FisherScientific) to the pre-weighed samples. The concentrations of sodium andpotassium were determined, using Inductively Coupled Plasma OpticalEmission Spectrometer (Varian 720-ES ICP OES, Australia).

Results

The concentration of sodium (Na⁺) and potassium ions (K⁺) in shoots isan important parameter for salt stress tolerance (Garriga, et al., JPlant Physiol, 210:9-17 (2017)). Therefore, the Na⁺ and K⁺ contents weredetermined in Arabidopsis organs in the absence and presence of SA187.Interestingly, both shoots and roots of SA187-inoculated plantsaccumulated similar levels of N⁺ compared with mock-inoculated plants,under normal and salt stress conditions (FIG. 3A, D). However, increasedK⁺ levels were found in SA187-colonized plants (FIG. 3B, E), resultingin significantly reduced shoot and root Na⁺/K⁺ ratios under salineconditions (FIG. 3C, F), which may help the inoculated plants to keephigh growth rate.

Example 4 Enterobacter sp. SA187 Colonizes Epidermis and Inner Tissuesof both Roots and Shoots

Methods

Generation of GFP-Labeled Bacteria

SA187 was genetically labeled with the GFP expressing cassette by takingadvantage of the mini-Tn7 transposon system (Choi, et al., Nat Methods,2:443-448 (2005)). In order to specifically select for a bacteriumcarrying the GFP integration in the genome, a spontaneous rifampicinresistant mutant of the strain was obtained first (Crotti, et al.,Environ Microbiol, 11:3252-3264 (2009)): an overnight-grown culture ofSA187 was plated on LB plates supplemented with 100 μg·mL⁻¹ ofrifampicin, and the plates were incubated for 24 h at 28° C. At least 10colonies, representing spontaneous rifampicin resistant mutants of thestrain were streaked twice on LB plates containing 100 μg·mL⁻¹ ofrifampicin and thereafter twice on LB plates supplemented with 200μg·mL⁻¹ of rifampicin. The GFP expressing cassette was introduced in theSA187 Rif^(R) strain by conjugation as described in Lambertsen et al.(Environ Microbiol, 6:726-732 (2004)). Briefly, 10¹⁰ cells of SA187Rif^(R) strain were mixed with 10⁹ cells of E. coli SM10γpir harboringthe helper plasmid pUX-BF13, the GFP donor (a mini-Tn7) plasmid andmobilizer pRK600 plasmid. The mixed culture was incubated on sterilenitrocellulose filter for 16 hrs. The conjugation culture of bacterialcells was resuspended in saline buffer (9 g/L NaCl) and spread onselective media with a propitiate antibiotics to select transformedSA187. The selected colonies were screened by fluorescence microscopyfor GFP fluorescence and positive colonies were further subjected togenotype confirmation by 16S rRNA gene sequencing.

Confocal Microscopy

GFP-labeled SA187 on Arabidopsis roots was imaged using an invertedZeiss LSM 710 confocal microscope equipped with Plan-Apochromat10×/0.45, Plan-Apochromat 20×/0.8, and Plan-Apochromat 40×/1.4 Oilobjectives. Seedlings grown for 3-21 days on vertical ½ MS agar platesor in soil inoculated with SA187-GFP were washed gently in steriledistilled water and transferred on a sterile agar plate. A block of agarwith several seedlings was immediately cut out and placed upside-down toa chambered cover glass (Lab-Tek™ II) with 30 μM propidium iodide (PI)in water as mounting medium. The GFP and PI fluorescence was excitedusing the 488 nm laser line, and captured as a single track (emission of493-537 nm for the GFP channel, 579-628 nm for the PI channel, 645-708nm for chloroplast autofluorescence). For 3D reconstructions, 1 μm-stepZ-stacks were taken, and images were generated in the integral 3D viewof the Zen software (Zeiss).

Quantification of Root Colonization

Col-0 seedlings were germinated on ½ MS agar plates, and transferred tonew ½ MS plates with or without 100 mM NaCl 5 days after germination (10seedlings per plate). Parts of their root systems grown after thetransfer were cut, gently washed by dipping in distilled water to removenon-attached bacterial cells, and then grinded in Eppendorf tubes usingteflon sticks. Each sample was resuspended in 1 ml of extraction buffer(10 mM MgCl₂, 0.01% Silwet L-77), sonicated for 1 min and subsequentlyvortexed for 10 min. Samples were diluted in 10× scale, spread on LBPetri dishes, and colonies were counted after overnight incubation at28° C. Calculated number of colony forming units was normalized percentimeter of root length (total root length was determined based onimages of root systems before their harvest). The experiment wasconducted in three biological replicates, each with three technicalreplicates per condition, each sample consisted of five roots.

Results

After recognition of the beneficial impact of SA187 on plant physiology,the interaction of SA187 with plants was characterized in more detail todetermine whether SA187 is able to efficiently colonize Arabidopsis asits non-native host. Therefore, its capacity to colonize Arabidopsisseedlings on ½ MS agar plates or in soil was analyzed by confocalmicroscopy. SA187 cells were stably transformed to express GFP(SA187-GFP), which did not affect their beneficial effect on Arabidopsisseedlings. Confocal microscopy revealed that SA187-GFP colonized bothroots and shoots on ½ MS agar plates or in soil (data not shown). Onvertical ½ MS agar plates, the first colonies (formed by a small numberof cells) were observed on the root epidermis in the elongation zone,preferentially in grooves between epidermal cell files (data not shown).In the differentiation zone and older root parts, colonies were largerand proportional with the age of the region (data not shown). A similarcolonization pattern was observed in soil-grown seedlings, however, witha more random distribution of colonies (data not shown). SA187-GFPcolonies were also often found in cavities around the base of lateralroots (data not shown). While it was rare to detect SA187-GFP cellsinside root tissues in 5-7 days old seedlings, the apoplast of the rootcortex and even of the central cylinder was regularly occupied by smallscattered colonies in 3 weeks old seedlings (data not shown). Indeed, inthe initial plant assays, SA187 could be re-isolated from surfacesterilized Arabidopsis roots, indicating that SA187 was proliferatinginside root tissues. Inspecting shoots, SA187-GFP colonies were alsofound deep inside the apoplast of hypocotyls, cotyledons and the firsttrue leaves, and in several cases bacterial cells were directly observedto penetrate through stomata of these organs (data not shown).

The colonization of root systems by SA187 (wild type strain) undernormal and salt conditions was examined. Plants were germinated on ½ MSagar plates containing SA187 wild type strains, transferred to new ½ MSplates with or without 100 mM NaCl after 5 days, and parts of their rootsystems grown after the transfer were used for bacterial extractionafter 5 more days. Interestingly, quantification based on counting ofcolony forming units (CFU) revealed that roots from salt conditions weretwice more colonized than those from normal conditions, suggesting thatin our experimental system plants can probably facilitate theiraccessibility to colonization by beneficial bacteria under stressconditions.

Example 5 SA187 Massively Reprograms Arabidopsis Gene Expression uponColonization

Methods

RNAseq and qPCR Analysis

Total RNA was extracted from 5-day-old plants either inoculated or notinoculated with SA187 and transferred for 10 more days on ½ MS plateswith or without 100 mM NaCl using the Nucleospin RNA plant kit(Macherey-Nagel), including DNasel treatment, and followingmanufacturer's recommendations.

RNA samples were analyzed by Illumina HiSeq deep sequencing (IlluminaHiSeq 2000, Illumina). Three biological replicates were processed foreach sample. Paired-end sequencing of RNA-seq samples was performedusing Illumina GAIIx with a read length of 100 bp. Reads werequality-controlled using FASTQC(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trimmomaticwas used for quality trimming (Bolger, et al., Bioinformatics,30:2114-2120 (2014)). Parameters for read quality filtering were set asfollows: Minimum length of 36 bp; Mean Phred quality score greater than30; Leading and trailing bases removal with base quality below 3;Sliding window of 4:15. TopHat v2.0.9 (Trapnell, et al., Bioinformatics,25:1105-1111 (2009)) was used for alignment of short reads to the A.thaliana genome TAIR10, Cufflinks v2.2.0 (Trapnell, et al., NatBiotechnol, 28:511-515 (2010)) for transcript assembly and differentialexpression. To identify differentially expressed genes, specificparameters (p-value: 0.05; statistical correction: Benjamini Hochberg;FDR: 0.05) in cuffdiff were used. Post-processing and visualization ofdifferential expression were done using cummeRbund v2.0.0 (Goff, et al.,(2001)). Gene was considered as regulated if fold change>log₂ ^(|0.6|)and q-value<0.05 compared to Mock condition. RNAseq data set can beretrieved under NCBI geo submission ID GSE102950.

For qPCR analysis, mock and SA187-inoculated plants were used for RNAextraction as described above. Samples were used for analysis of eitherplant or SA187 gene expression. For bacteria alone, SA187 incubated for4h in liquid ½ MS or ½ MS with 100 mM NaCl at 28° C. and dark were usedfor RNA extraction, using the RiboPure™ RNA Purification Kit (Ambion),following manual instructions for Gram-negative bacteria, with theexception that no beads were added during bacterial lysis. RNAextraction was followed by DNAseI treatment.

cDNAs were synthetized using SuperscriptIII (Invitrogen): 1 μg of totalRNA, oligo-dT as a primer, following manufacturer's recommendations. ForArabidopsis gene expression analyses, ACTIN2 (At3g18780) and UBIQUITIN10(At4g05320) were used as reference genes. For SA187 gene expressionanalyses, infB, rpoB and gyrB were used as reference genes. Allreactions were done in a CFX96 Touch™ Real-Time PCR Detection System(BIO-RAD) as follows: 50° C. for 2 min, 95° C. for 10 min; 40×[95° C.for 10 sec and 60° C. for 40 sec]; and a dissociation step to validatePCR products. All reactions were performed in three biologicalreplicates, and each reaction as a technical triplicate. Gene expressionlevels were calculated using the Bio-Rad CFX manager software. Primersequences used in this analysis are listed in Table 1 below.

TABLE 1 Primers Sequences Used. Primer name Sequence (5′ to 3′)Targetted gene Purpose of the primer 27F AGAGTTTGATCCTGGCTCAG 16S rRNABacterial strain identification (SEQ ID NO: 14) 1492RTACGGYTACCTTGTTACGACTT (SEQ ID NO: 15) P922-3G56400F1TGGTTCGTCCACGGAGAATG AT3G56400 qPCR analysis (SEQ ID NO: 16)P923-3G56400R1 CCCATTGACGTAACTGGCCT (SEQ ID NO: 17) P854-1G01580F1GCAAGCGAAGCTGGAATCAG AT1G01580 qPCR analysis (SEQ ID NO: 18)P855-1G01580R1 AATCCCATTGCCGGTAGCAA (SEQ ID NO: 19) P926-4G01250F1ACTAGCAAACCCAGTGGCTC AT4G01250 qPCR analysis (SEQ ID NO: 20)P927-4G01250R1 CATGCCCAGACATCGGAGTT (SEQ ID NO: 21) P888-1G80440F2ATCGCTACGCCTGAATACCG AT1G80440 qPCR analysis (SEQ ID NO: 22)P889-1G80440R2 CCAGGAATCGGAGGAAGCTC (SEQ ID NO: 23) P866-1G27730F1AGTCGAGCACTGGACAAAGG AT1G27730 qPCR analysis (SEQ ID NO: 24)P867-1G27730R1 TAGCTCAACTTCTCCACCGC (SEQ ID NO: 25) P245-AT2G37870F1CTGTGCCAAAGTTGGTGCTC AT2G37870 qPCR analysis (SEQ ID NO: 26)P246-AT2G37870R1 GTAACGTCCACATCGCTTGC (SEQ ID NO: 27) P930-4G13420F1TACGTGGGGCCAAAGGATTC AT4G13420 qPCR analysis (SEQ ID NO: 28)P931-4G13420R1 CCCTCCTCCTCCAGACATGA (SEQ ID NO: 29) P309-AT3G30775F1CAACCCGTCTTCTCCGAACA AT3G30775 qPCR analysis (SEQ ID NO: 30)P310-AT3G30775R1 CGGTGCTTGTTGTCCAAAGG (SEQ ID NO: 31) P237-AT3G53980F1CCGTCGGTTACAAGTGTGGA AT3G53980 qPCR analysis (SEQ ID NO: 32)P238-AT3G53980R1 AGGCCCAATGTTATCTCCTTCA (SEQ ID NO: 33) P329-AT4G37800F1TTGGTTCGACCCTTCTCGTG AT4G37800 qPCR analysis (SEQ ID NO: 34)P330-AT4G37800R1 CCCTGATGGGCACATTGTCT (SEQ ID NO: 35) P870-1G35140F1TGGATGCGAGAACGGACAAA AT1G35140 qPCR analysis (SEQ ID NO: 36)P871-1G35140R1 CATGGTCGATCTCCGGGAAG (SEQ ID NO: 37) P281-AT2G39030F1GAGTCTGGTCTTGCCTCCAC AT2G39030 qPCR analysis (SEQ ID NO: 38)P282-AT2G39030R1 ATGCGTCTCAAGAAAGGGGG (SEQ ID NO: 39) P962-5G51190F1CCAACGCAAACCACCTCTTG AT5G51190 qPCR analysis (SEQ ID NO: 40)P963-5G51190R1 CAGCCGCATACTTACCCCAT (SEQ ID NO: 41) P862-1G13260F1GGTGTTTCTACGACGGGGTT AT1G13260 qPCR analysis (SEQ ID NO: 42)P863-1G13260R1 TTAGCTTCCCAACGTCGCTT (SEQ ID NO: 43) P934-4G17490F1GGCGATTCTGAATTTCCCGC AT4G17490 qPCR analysis (SEQ ID NO: 44)P935-4G17490R1 TTGTACAGGCCACGACCATC (SEQ ID NO: 45) P882-1G74930F1CTTTCGACGCCGCTCAATTT AT1G74930 qPCR analysis (SEQ ID NO: 46)P883-1G74930R1 GGAGGCGTCAACGACTTTTC (SEQ ID NO: 47) P918-3G55980F1ATTGCAGACGTGTCGGTTCT AT3G55980 qPCR analysis (SEQ ID NO: 48)P919-3G55980R1 CAGCACAGTGAAGCGGAGTA (SEQ ID NO: 49)

Hierarchical Clustering and Gene Family Enrichment

Arabidopsis regulated genes were used to generate HCL tree using MultiExperiment Viewer (MeV 4.9.0 version, TM4. Raw data were normalized forevery gene. Hierarchical clustering was performed using Euclidiandistances, average linkage and leaf order optimization.

Gene enrichment analyses were performed using AmiGO website.

Results

To uncover how salt stress tolerance is achieved in SA187-inoculatedArabidopsis seedlings, RNA-seq analysis was performed to compare thetranscriptome of mock-inoculated to SA187-inoculated plants undernon-saline (Mock and SA187), and salt stress conditions (Salt andSA187+Salt). Compared to mock conditions, 545, 3113 and 1822 genes werefound to be differentially expressed in the “SA187”, “Salt” and“SA187+Salt” samples, respectively. To obtain a global overview, thetranscriptome data were organized by hierarchical clustering into 8groups and analyzed for gene ontology enrichment (FIG. 4A-F).

Cluster 1 and 7 comprise the largest sets of differentially expressedgenes with 1607 and 744 members, respectively, and consist ofsalt-stress regulated genes that were unaffected by the SA187inoculation (FIG. 4B and 5F). Whereas Cluster 1 genes are stronglydownregulated under salinity and are involved in water homeostasis,salicylic acid (SA) and defense response, those of Cluster 7 are highlyupregulated and enriched in genes that are induced in response to waterand salt stress or abscisic acid (ABA).

A specific effect of SA187 on the transcriptome of plants was found inClusters 2, 3 and 4 (FIGS. 4C-E). Cluster 2 (354 genes) represents genesthat are upregulated by SA187 independently of the growth conditions(FIG. 4C). This cluster was significantly enriched in plant defensegenes such as chitin responsive genes but also in ethylene and jasmonicacid (JA) signaling (FIG. 4A-F). Importantly, Cluster 3 genes (246) arestrongly downregulated in mock-inoculated plants under salt stressconditions but remain unaltered upon SA187-inoculation (FIG. 4D). Thesegenes have a role in the primary metabolism, such as photosynthesis,carbon and energy metabolisms. On the contrary, Cluster 4 genes (464)are enriched in ABA and abiotic stress response and are upregulated insalt-treated plants, but not when the plants were inoculated with SA187(FIG. 4E).

In summary, these data indicate that SA187 colonization triggers inArabidopsis the expression of genes involved in defense response asshown by the significant enrichment for chitin responsive genes andethylene and JA signaling. Moreover, under saline conditions,SA187-inoculated plants release themselves from the impact of abioticstress (ABA), maintain higher metabolic and photosynthetic activity, andcan therefore grow better than mock-inoculated plants.

Example 6 SA187 modulates Abscisic Acid, Jasmonic Acid, and EthyleneHormonal Pathways under Salt Stress

Methods

Hormone Content Analysis

For each sample, 10 mg of freeze-dried powder were extracted with 0.8 mLof acetone/water/acetic acid (80/19/1 v:v:v). For each sample, 2 ng ofeach standard was added to the sample: abscisic acid, salicylic acid,jasmonic acid, and indole-3-acetic acid stable labeled isotopes used asinternal standards were prepared as described previously (Le Roux, etal., PLoS One, 9:e99343 (2014)). The extract was vigorously shaken for 1min, sonicated for 1 min at 25 Hz, shaken for 10 minutes at 4° C. in aThermomixer (Eppendorf), and then centrifuged (8000 g, 4° C., 10 min).The supernatants were collected, and the pellets were re-extracted twicewith 0.4 mL of the same extraction solution, then vigorously shaken (1min) and sonicated (1 min; 25 Hz). After the centrifugations, threesupernatants were pooled and dried.

Each dry extract was dissolved in 140 μL of acetonitrile/water (50/50;v/v), filtered, and analyzed using a Waters Acquity ultra performanceliquid chromatograph coupled to a Waters Xevo Triple quadrupole massspectrometer TQS (UPLC-ESI-MS/MS). The compounds were separated on areverse-phase column (Uptisphere C18 UP3HDO, 100×2.1 mm, 3 μm particlesize; Interchim, France) using a flow rate of 0.4 mL·min⁻¹ and a binarygradient: (A) acetic acid 0.1% in water (v/v) and (B) acetonitrile with0.1% acetic acid. For ABA, salicylic acid, jasmonic acid, the followingbinary gradients were used (time, % A): (0 min, 98%), (3 min, 70%), (7.5min, 50%), (8.5 min, 5%), (9.6 min, 0%), (13.2 min, 98%), (15.7 min,98%), and the column temperature was 40° C. Mass spectrometry wasconducted in electrospray and Multiple Reaction Monitoring scanning mode(MRM mode), in the negative ion mode. Relevant instrumental parameterswere set as follows: capillary 1.5 kV (negative mode), source block anddesolvation gas temperatures 130° C. and 500° C., respectively. Nitrogenwas used to assist the cone and desolvation (150 L·h⁻¹ and 800 L·h⁻¹,respectively), argon was used as the collision gas at a flow of 0.18mL·min⁻¹. Samples were reconstituted in 140 μL of 50/50 acetonitrile/H₂O(v/v) per mL of injected volume. The limit of detection (LOD) and limitof quantification (LOQ) were extrapolated for each hormone fromcalibration curves and samples using Quantify module of MassLynxsoftware, version 4.1.

GUS Staining

Seedlings were vacuum infiltrated with the pre-fixation buffer [0.3%formaldehyde, 0.28% mannitol, 50 mM sodium phosphate buffer (pH 7.2)],washed with phosphate buffer and incubated in staining solution [250 μMK₃Fe(CN)₆, 250 μM K₄Fe(CN)₆, 2% Triton-X, 1 mM5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid (X-GlcA; Duchefa), 50 mMsodium phosphate buffer (pH 7.2)]. Tissue was cleared with Visokol(Phytosys) overnight and observed with Axio Imager 2 (Zeiss) equippedwith Plan-Neofluar 10×/0.45 objective.

Results

Since the transcriptome analysis indicated possible roles of severalhormone pathways in the SA187-induced growth promotion under saltstress, the levels of salicylic acid (SA), jasmonic acid (JA) andabscisic acid (ABA) were measured in mock- and SA187-inoculated plants.SA187 did not significantly change plant SA levels in the absence orpresence of salt (FIG. 5A). Plant ABA and JA concentrations alsoremained unchanged upon SA187 colonization under normal conditions, buttheir salt-induced accumulation was significantly lower inSA187-inoculated plants (FIG. 5B-C), indicating a partial attenuation ofstress responses in these plants.

To assess the level of ethylene in Arabidopsis roots and possiblyconfirm the activation of the ethylene signaling pathway observed inCluster 2, the ethylene-dependent pEBF2::GUS reporter was used (Konishi,et al., Plant J, 55:821-831 (2008)). In contrast to mock-inoculatedseedlings (FIG. 5D), the reporter line showed strong GUS activity inroot tips upon SA187-inoculation (FIG. 5E), similar to the treatmentwith the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC)(FIG. 5F), indicating the activation of the ethylene signaling pathway.

Example 7 Ethylene Perception Mutants are Compromised in the BeneficialResponse to SA187

To substantiate the phytohormone quantifications, Arabidopsis hormonedeficient or insensitive mutants were analyzed. The JA-receptor coil-1mutant (Xie, et al., Science, 280:1091-1094 (1998)), JA-insensitivejarl-1 (Staswick, et al., Proc Natl Acad Sci USA, 89 :6837-6840 (1992)),the ABA biosynthesis aba2-1 mutant (Schwartz, et al., Plant Physiol, 114:161-166 (2997)), or the ABA receptor quadruple pyr1-1pyl1-1pyl2-1pyl4-1mutant (Park et al., Science, 324:1068-1071 (2009)) maintained the SA187beneficial activity upon salt stress, indicating that ABA or JA may notplay a major role in this interaction (FIG. 6A-B).

However, the ethylene insensitive ein2-1 and ein3-1 mutants (Guzman, etal., Plant Cell, 2:513-523 (1990); Roman, et al., Genetics,139:1391-1409 (1995)), impaired in ethylene perception, were stronglycompromised in the beneficial effect of SA187, indicating that ethylenesensing could be of importance in SA187-induced tolerance of Arabidopsisto salt stress conditions. This result was confirmed by theup-regulation of the four ethylene-induced genes, ERF105 (FIG. 6D),ERF018 (FIG. 6F), RAV1 (FIG. 6E) and SZF1 (FIG. 6G), upon colonizationby SA187. Moreover, application of 100 nM ACC during salt stress couldlargely mimic the beneficial activity of SA187 on plants (FIG. 6C).

In contrast, the heptuple ethylene-biosynthesis deficient mutant acs1-1acs2-1 acs4-1 acs5-2 acs6-1 acs?-1 acs9-1 (called acs in this study)still showed full sensitivity to the beneficial activity of SA187 undersalt stress (FIG. 6A). Additionally, the SA187 beneficial effect wasmaintained when plants were treated with amino-ethoxy-vinyl glycine(AVG, 1 μM), an ethylene production inhibitor blocking ACC synthesis(Schaller, et al., in: Ethylene Signaling Methods and Biosynthesis, pp233-235 (2017)) (FIG. 6H). However, when plants were treated with silvernitrate (AgNO₃, 1 μM), which interferes with ethylene perception(Schaller, et al., in: Ethylene Signaling Methods and Biosynthesis, pp233-235 (2017)), inoculated plants did not exhibit any SA187-inducedtolerance to salt stress (FIG. 6H).

Altogether, these results indicate that the beneficial effect of SA187may not be mediated by JA perception or the ABA pathway, but rather byethylene perception, as it was found to be necessary for SA187-inducedsalt stress tolerance on Arabidopsis plants.

Example 8 Arabidopsis Upregulates the Methionine Salvage Pathway inSA187

Methods

Measurement of in vitro Ethylene Emanation

A fresh SA187 culture was prepared by inoculation of 50 mL of liquid LBmedium with 1 mL of overnight-grown culture. Subsequently, 2 mL of freshculture was transferred to 10 mL chromatography vials and sealed with arubber plug and snap-cap (Chromacol) after 0, 1, 2 or 4 hours of growthon a shaker incubator (220 rpm, 28° C.). The sealed vials were againtransferred to the shaker incubator for another 2 hours to allowethylene accumulation. Three biological replicates were prepared at eachtime point along with 3 controls to correct for background ethyleneemanation. Ethylene emission was measured with a laser-basedphoto-acoustic detector (ETD-300 ethylene detector, Sensor Sense, TheNetherlands) (van de Poel, et al., Methods in Molecular Biology:Ethylene Signaling, Methods and Protocols, 2017). Immediately after theethylene measurement, OD₆₀₀ was determined with Implen NanoPhotometerNP80 (Sopachem Life Sciences, Belgium) to correct for the total amountof bacterial cells present in the samples.

Results

The previous results suggested that ethylene most likely originates fromSA187 cells rather than from the canonical plant ACC synthase (ACS)pathway. To support the hypothesis that SA187 provides ethylene topromote plant growth, the genome of SA187 was searched for bacterialgenes encoding ACS or ethylene forming enzymes (EFE). No ACS- orEFE-related genes were found in SA187, but the methionine salvagepathway is conserved in SA187, and one of its components, KMBA, is knownto be an ethylene precursor (Eckert, et al., Biotechnol Biofuels, 7:33(2014)). While SA187 alone did not produce ethylene when grown onsynthetic media, the expression level of most of the genes encoding theKMBA pathway were upregulated in SA187 upon plant colonization comparedwith bacteria incubated for 4h in liquid ½ MS with or without 100 mMNaCl in the absence of plants (FIG. 7A-B).

To confirm that KMBA could function as an ethylene precursor during thebeneficial plant-microbe interaction, the effect of KMBA on Arabidopsisin comparison to SA187 inoculation was tested. Under salt stressconditions, application of 100 nM KMBA induced a similar beneficialactivity on Arabidopsis as SA187 with a similar increase in both rootand shoot fresh weight (FIG. 7C).

2,4-dinitrophenylhydrazine (DNPH) is a known interactor of KMBA in vitrothat was previously shown to precipitate Botrytis cinerea produced KMBAand consequently impair the production of ethylene by photo-oxidation(Chague, et al., FEMS Microbiol Ecol, 40:143-149 (2002)). When plantswere cultivated with 3 μM DNPH, SA187-induced salt tolerance was greatlyreduced from 68% to 14% (FIG. 7D-E), showing the importance of KMBA inmediating SA187-induced plant tolerance to salt stress.

While in the foregoing specification this invention has been describedin relation to certain embodiments thereof, and many details have beenput forth for the purpose of illustration, it will be apparent to thoseskilled in the art that the invention is susceptible to additionalembodiments and that certain of the details described herein can bevaried considerably without departing from the basic principles of theinvention.

All references cited herein are incorporated by reference in theirentirety. The present invention may be embodied in other specific formswithout departing from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indicating the scope of theinvention.

We claim:
 1. A method for increasing salt tolerance in a plantcomprising: treating the plant with an amount of2-keto-4-methylthiobutyric acid effective to increase salt tolerance inthe plant relative to an untreated plant.
 2. The method of claim 1,wherein the amount of 2-keto-4-methylthiobutyric acid is in solution. 3.The method of claim 1, wherein the solution is an aqueous solution. 4.The method of claim 1, wherein the amount of 2-keto-4-methylthiobutyricacid is in water.
 5. The method of claim 2, wherein the amount of2-keto-4-methylthiobutyric acid is in salt water and optionally, whereinthe solution comprises seawater.
 6. The method of claim 2, wherein thesolution comprises 1.0 to 10% salt, optionally, wherein the salt isselected from the group consisting of salts of sodium, chloride,sulfate, magnesium, calcium, potassium, and combinations thereof.
 7. Themethod of claim 1 further comprising inoculating the plant with amicrobial system to produce KMBA in the plant.
 8. The method of claim 6,wherein the solution comprises 3.5% salt.
 9. The method of claim 7,wherein the microbial system to produce KMBA in the plant comprises aneffective amount of SA187 to colonize plant seed or a root of the plantto provide abiotic stress resistance to the seed or plant; or abacterium genetically engineered to overexpress express L-amino aciddeaminase (L-AAD) from Proteus vulgaris or one or more genes from themethionine salvage pathway selected from the group consisting of mtnN(EC 3.2.2.16), tyrB encoding a wide spectrum aminotransferase, mtnA,encoding MtnA (methylthioribose-1-phosphate isomerase), mtnB, encoding amethylthioribulose-1-phosphate dehydratase; mtnC, encoding anenolase-phosphatase, mtnD (ykrZ) , encodiong an aci-reductonedioxygenase; mtnK, encoding a methylthioribose kinase; metK, enclodingS-adenosylmethionine (SAM) synthetase; speE encoding a spermidinesynthetase; speD, encoding an S-adenosylmethionine, decarboxylase.
 10. Amethod for cultivating a plant comprising: planting seeds or seedlingsof the plant; and irrigating the seeds or seedlings with a salinesolution comprising 2-keto-4-methylthiobutyric acid.
 11. The method ofclaim 1, wherein the plant is a forage crop.
 12. The method of claim 10,wherein the plant is selected from the group consisting of rice, wheat,sugarcane, maize, soybean, cotton, vegetables, rape, mustard, sorghum,millet, grass, Brassica spp., rapeseed, barley, hay, and alfalfa.
 13. Amethod of increasing tolerance to abiotic stress in a plant comprising:treating the plant with a composition comprising an effective amount of2-keto-4-methylthiobutyric acid to increase tolerance to the abioticstress.
 14. The method of claim 13, wherein the abiotic stress isselected from the group consisting of water salinity, soil salinity, orboth, and optionally, growing the seed or plant in a plant substrate,wherein the plant substrate comprises an effective amount of SA187 tocolonize the seed or a root of the plant to provide abiotic stressresistance to the seed or plant.
 15. The method of claim 1, wherein the2-keto-4-methylthiobutyric acid is present at a concentration of 1.0 to250 nM.
 16. The method of claim 15, wherein the2-keto-4-methylthiobutyric acid is present at a concentration of atleast 100 nM.
 17. A method of cultivating a plant in sodic soilcomprising: contacting the plant in sodic soil with an effective amountof 2-keto-4-methylthiobutyric acid to increase salt tolerance in theplant.
 18. An irrigation solution comprising: 1.0 to 250 nM2-keto-4-methylthiobutyric acid in an aqueous saline solution.
 19. Thesolution of claim 18, wherein the aqueous saline solution is seawater.20. The irrigation solution of claim 18, wherein the aqueous salinesolution comprises 1 to 10% salt.